Fluorescent conjugates of nucleosides or nucleotides, process for their preparation and their uses

ABSTRACT

The invention relates to novel fluorescent conjugates of nucleosides or nucleotides which can be used especially for detecting, locating and/or isolating nucleic acids or molecules of biological or clinical interest which have a nucleoside structure or are capable of interacting with nucleic acids. The invention further relates to the polynucleotides comprising at least one fluorescent conjugate of a nucleotide.

This is a 371 of PCT/FR98/02111 filed on Oct. 2, 1998.

The invention relates to novel fluorescent conjugates of nucleosides ornucleotides which can be used especially for detecting, locating and/orisolating nucleic acids or molecules of biological or clinical interestwhich have a nucleoside structure or are capable of interacting withnucleic acids.

The following abbreviations will be used in the remainder of thedescription:

RNA: ribonucleic acid

DNA: deoxyribonucleic acid

A: adenosine

C: cytidine

G: guanosine

T: thymidine

U: uridine

I: inosine

suffix MP: monophosphate

suffix DP: diphosphate

suffix TP: triphosphate

prefix d: deoxy

A reaction for the enzymatic synthesis of DNA employs an RNA or DNAtemplate, an oligonucleotide primer whose sequence is complementary to asegment of the template, an appropriate enzyme, and the fourdeoxynucleotides dATP, dCTP, dGTP and dTTP (or dUTP). Various enzymesare known, such as E. coli DNA polymerase, T7 DNA polymerase, the Klenowfragment of DNA polymerase, Taq DNA polymerase and a reversetranscriptase, to which may be added terminal nucleotidyl transferase,which has the particular characteristic of not requiring a template. Thesynthesis of RNA is carried out in a similar manner except that therequired primers and templates are different and ribonucleotides (ATP,CTP, GTP and UTP) are used in the presence of RNA polymerases.

Nucleotides or polynucleotides can be labeled radioactively (³H, ³²P,¹⁴C, ³⁵S or ¹²⁵I).

The use of labeled molecules has the disadvantages conventionallyassociated with radioactive isotopes, namely the risks inherent inradioactivity as well as the limited storage and availability due toradioactive decay and radiolysis phenomena.

The chemical labeling of nucleotides or polynucleotides, which makes itpossible to avoid these disadvantages, has been described in theliterature. The biotin labeling of nucleotides derived from dUTP or UTPhas been described in particular (Langer P. R., Waldrop A. A., Ward D.C., 1981, Proc. Natl. Acad. Sci. USA, 78, 6633-37). These arederivatives in which the biotin is bonded to the C-5 position of thepyrimidine ring by an alkylamine arm. These labeled nucleotides areincorporated in vitro into polynucleotides by the action of DNA or RNApolymerases (EP 0 063 879) and allow the calorimetric detection ofnucleic acids by means of a dot-blot reaction (Leary J. J., Brigati D.J. and Ward D. D., 1983, Proc. Natl. Acad. Sci. USA, 80, 4045-49).

Other analogs of biotin-labeled nucleotides, based on derivatives ofN-4-aminoalkyldeoxycytidine and N-6-aminoalkyldeoxyadenosine, aredescribed in patent U.S. Pat. No. 4,828,979 and in Gebegehu G. G. etal., Nucleic Acids Res., 1987, 15, 4513-4534.

Derivatives of dUTP and of dATP substituted by biotin in the C-8position, as well as the possibility of the C-7 substitution of a7-deazapurine, are also described (EP 0 063 879).

The derivative bio-15-dGTP has also been described (Gilliam I. C. andTener G. M., 1989, Nucleoside & Nucleotide, 8, 1453-62). Fluorescentderivatives such as fluorescein or rhodamine can be incorporated into anucleic acid via a labeled nucleoside triphosphate (dATP, dUTP, dCTP)(WO 93 19206). A discussion about the position of substitutions onpurines and pyrimidines and about the nature of the spacer arms whichcan be used to label dideoxynucleotides with fluorescein derivatives,although devoted to chain terminators for sequencing, gives an overviewof the chemistry of labeled nucleotides (Confalone P. T., 1990, J.Heterocyclic Chem., 27, 31-46).

The combined use of nucleoside triphosphates labeled with differenttracers (biotin-11-dUTP, dig-11-dUTP and FITC-11-dUTP) affords thesimultaneous visualization of different sequences during a hybridization(REED T. et al., Proc. Natl. Acad. Sci. USA (1992), 89, 1388-1392).

The incorporation of fluorescein-labeled deoxynucleotides such asFl-dUTP or Fl-dCTP is also effected by replacing only part of thenatural nucleotide with the labeled compound: dCTP/Fl-dCTP≈2:1 (WO93/19206). This same patent describes that, by choosing the enzyme andthe experimental conditions, it is possible to replace the whole of anucleotide with a labeled nucleotide.

The literature data show that the efficiency of incorporation of atripbosphate conjugate is modified by coupling with a molecule such asbiotin and to a lesser extent by the presence of the arm which allowsthe coupling.

For example, in a nick translation reaction in the presence of a DNApolymerase, the degrees of incorporation of various triphosphate analogswere compared with the natural nucleoside taken as a reference (100%incorporation) for the same reaction time (90 min) (Gebeyehu G. et al.,Nucleic Acids Res., 1987, 15, 4525).

Molecules conjugated with nucleoside triphosphates (Goodchild, J.Bioconjugate Chem., 1990, p. 171; Kricka J., Non isotopic Blotting, andSequencing, 1995, Academic Press, p. 47; Zhu Z. et al., Nucleic AcidsRes., 1994, 3418-3422) are molecules of relatively small size (<800 Da)which are either neutral or negatively charged and have an essentiallyplanar shape; consequently, their bulk is reduced but sufficient todisturb the enzymatic incorporation of the nucleoside triphosphate.

Novel conjugates of nucleosides or nucleotides have now been found whichcomprise:

a ribo- or deoxyribo-nucleoside or -nucleotide which is native,chemically modified or conjugated with one or more labeling molecules,in which at least one carbon atom of the ring, exocyclic nitrogen atomof the purine or pyrimidine ring or carbon atom of the pentofuranosering may be involved in bonding with a fluorescent marker, and

at least one fluorescent marker bonded to said atom(s) and consisting ofa rare earth cryptate.

The invention may be better understood from the following detaileddescription, taken in conjunction with the accompanying drawings, inwhich:

FIG. 1 illustrates a plot of the value of DF as a function of reactiontime for incorporation of K-11-dUTP;

FIG. 2 illustrates the chemical formulae of K-4-dUTP and K-11-dUTP;

FIG. 3 illustrates the chemical formulae of K-11-UTP and K-9-ATP; and

FIG. 4 illustrates a plot of the value of DF as a function of reactiontime for incorporation of K-11-UTP.

According to a preferred feature, said conjugates comprise:

a ribo- or deoxyribo-nucleoside or -nucleotide which is native,chemically modified or conjugated with one or more labeling molecules,in which at least one carbon atom of the ring or exocyclic nitrogen atomof the purine or pyrimidine ring may be involved in bonding with afluorescent marker, and

at least one fluorescent marker bonded to said atom(s) and consisting ofa rare earth cryptate.

Said conjugates can be used in any applications of labeled nucleosidesor nucleotides without their use presenting major disadvantages, and inparticular they have a high capacity for incorporation intosingle-stranded or double-stranded DNA.

These properties are all the more surprising because rare earthcryptates are molecules of high molecular weight (greater than 1400 Da),possess a three-dimensional structure exhibiting more steric hindrancethan a globally plane molecule, and are of an ionic nature due to thepresence of the complexed ion, which gives them a positive charge.

Given these structural characteristics, the positive charges of a rareearth cryptate could be expected to interact strongly with the negativecharges of the triphosphate group and to modify its reactivity as wellas the fluorescent properties of the cryptate.

Also, as the activity of enzymes such as polymerases is sensitive to thepresence and concentration of certain complexing ions or agents in thereaction medium, the conjugates according to the invention could beexpected to cause a considerable decrease in the incorporation of thenucleotide triphosphates, and hence a low yield, especially during apolynucleotide synthesis.

Surprisingly, the results obtained using the conjugates according to theinvention show that not only do rare earth cryptates not have anunfavorable influence in applications involving enzymatic reactions, butalso they retain their intrinsic fluorescent properties.

Advantageously, it has also been found that the conjugates according tothe invention have novel fluorescent characteristics and that, inparticular, the emission lifetime of the complexed rare earth ion issignificantly increased.

Said conjugates can therefore be used as fluorescent markers in any useswhere a quantitative or qualitative detection is effected by measurementof the direct or indirect fluorescence.

According to a preferred feature, the invention relates to fluorescentconjugates of nucleotides comprising a ribonucleotide selected from AMP,ADP, ATP, GMP, GDP, GTP, CMP, CDP, CTP, UMP, UDP, UTP, TMP, TDP, TTP,2Me-AMP, 2Me-ADP, 2Me-ATP, 1Me-GMP, 1Me-GDP, 1Me-GTP, 5Me-CMP, 5Me-CDP,5Me-CTP, 5MeO-CMP, 5MeO-CDP, 5MeO-CTP, 7-deaza-ATP and 7-deaza-GTP, or,if appropriate, a deoxyribonucleotide selected from the deoxy- ordideoxyribonucleotides corresponding to these ribonucleotides, and inparticular:

2′-deoxyuridine 5′-triphosphate or uridine 5′-triphosphate derivativesfunctionalized in the 5-position of the base (principally an aminoallylor aminopropyne skeleton),

2′-deoxycytidine 5′-triphosphate or cytidine 5′-triphosphate derivativesfunctionalized in the 4- or 5-position of the base (principally anaminoallyl or aminopropyne skeleton in the case of the 5-position),

2′-deoxyadenosine 5′-triphosphate or adenosine 5′-triphosphatederivatives functionalized in the 6- or 8-position of the base,

2′-deoxyguanosine 5′-triphosphate or guanosine 5′-triphosphatederivatives functionalized in the 6- or 8-position of the base,

2′-deoxy-7-deazaadenosine 5′-triphosphate or 7-deazaadenosine5′-triphosphate derivatives functionalized in the 7-position of thebase, and

2′-deoxy-7-deazaguanosine 5′-triphosphate or 7-deazaguanosine5′-triphosphate derivatives functionalized in the 7-position of thebase.

The nucleotides which can be used for the purposes of the invention alsoinclude nucleotides chemically modified on the triphosphate part,particularly α-thiotriphosphate derivatives.

According to another feature, the invention relates to fluorescentconjugates of nucleosides in which the ribo- or deoxyribonucleoside isselected from A, G, C, U, T, the corresponding deoxy- ordideoxynucleosides and their chemically modified analogs, particularly3′-azido-3′-deoxythymidine and its derivatives, and the 2′,3′-dideoxyanalogs of A, T, C, G, U and I.

The fluorescent marker consists of a rare earth cryptate preferablyselected from terbium, europium, samarium or dysprosium cryptates.

In the remainder of the description, the term “cryptate” and thenomenclature of the macrocycles and polycycles which can be used are asdefined by J. M. Lehn in Struct. Bonding (Berlin), 16, 1, 1973 and inAcc. Chem. Res., 11, 49 (1978).

According to a preferred feature, said rare earth cryptate consists ofat least one rare earth salt complexed by a macropolycyclic compound ofthe formula

in which Z is a tri- or tetravalent atom, R is nothing, hydrogen, thehydroxyl group, an amino group or a hydrocarbon radical, and thedivalent radicals Â, {circle around (B)} and Ĉ independently of oneanother are hydrocarbon chains which optionally contain one or moreheteroatoms and are optionally interrupted by a heteromacrocycle, atleast one of the radicals Â, {circle around (B)} and Ĉ also containingat least one molecular moiety or essentially consisting of a molecularmoiety, said molecular moiety possessing a greater triplet energy thanthat of the emission level of the complexed rare earth ion.

Said rare earth cryptate is preferably a cryptate of formula (I) abovein which the molecular moiety is selected from phenanthroline,anthracene, benzene, naphthalene, bi- and terphenyl, azobenzene,azopyridine, pyridine, bipyridines, bis-quinolines and the compounds ofthe following formulae:

—C₂H₄—X₁—C₆H₄—X₂—C₂H₄—

—C₂H₄—X₁—CH₂—C₆H₄—CH₂—X₂—C₂H₄—

in which X₁ and X₂, which can be identical or different, are oxygen,nitrogen or sulfur, and

in which X is oxygen or hydrogen.

According to an advantageous feature, the fluorescent marker is a rareearth cryptate consisting of the terbium or europium ion complexed byone of the following macrocyclic compounds:

[2.2.phenanthroline]; [2.2.phenanthroline amide]; [2.2.anthracene];[2.2.anthracene amide]; [2.2.biisoquinoline];[2.2.biphenyl-bis-pyridine]; [2.2.bipyridine]; [2.2.bipyridine amide];and tris-bipyridine, tris-phenanthroline, phenanthroline-bisbipyridine,biisoquinoline-bis-bipyridine and bis-bipyridine diphenylbipyridinemacropolycycles.

One particularly advantageous marker is the europium cryptate Eutris-bipyridine.

Such compounds are described for example in patent EP 180 492.

It is also possible to use macropolycyclic cryptate compounds whichcomplex rare earth ions and in which the molecular moiety is selectedfrom bipyrazines, bipyrimidines and nitrogen heterocycles containingN-oxide groups.

Macropolycyclic compounds with bipyrazine units are described in F.Bodar-Houillon et al., New J. Chem., 1996, 20, 1041-1045.

Macropolycyclic compounds with bipyrimidine units are described in J. M.Lehn et al., Helv. Chim. Acta, 1992, 75, 1221.

Macropolycyclic compounds comprising nitrogen heterocycles containingN-oxide groups are described in J. M. Lehn et al., Helv. Chim. Acta,1991, 74, 572.

The rare earth cryptate used as the fluorescent marker can also consistof at least one rare earth salt complexed by a macropolycyclic compoundof formula II or III below:

in which:

the ring of the formula

 is one of the following rings:

Y is a group or spacer arm consisting of a divalent organic radicalselected from linear or branched C₁ to C₂₀ alkylene groups optionallycontaining one or more double bonds and/or optionally containing one ormore heteroatoms such as oxygen, nitrogen, sulfur or phosphorus, or oneor more carbamoyl or carboxamido groups, from C₅ to C₈ cycloalkylenegroups or from C₆ to C₁₄ arylene groups, said alkylene, cycloalkylene orarylene groups optionally being substituted by alkyl, aryl or sulfonategroups;

Z is a functional group capable of bonding covalently with a biologicalsubstance;

R is a methyl group or the group -Y-Z; and

R′ is hydrogen or a group —COOR″, in which R″ is a C₁ to C₁₀ alkylgroup, preferably the methyl, ethyl or tert-butyl group, or R′ is agroup —CO—NH—Y—Z.

Such compounds are described for example in patent EP 321 353.

Within the conjugates according to the invention, said fluorescentmarker can be bonded to the ribo- or deoxyribo-nucleoside or -nucleotideeither directly or via a spacer arm.

“Direct bonding” is understood as meaning the bonding of the fluorescentmarker to a functional group previously introduced onto or generated onone or more atoms of the base or of the pentofuranose unit of the ribo-or deoxyribonucleoside or -nucleotide.

In the present description, functional group is understood as meaningany function carried by the nucleoside or nucleotide part or introducedonto this part by any method known to those skilled in the art, andcapable of bonding covalently, either directly or after activation, witha function present on the cryptate or on the spacer arm carried by thecryptate. Such functional groups are especially the NH₂, COOH, CHO, OHor SH functions, as well as functions capable of producing covalentbonds by substitution (halides, sulfonates, epoxide) or by addition(double bonds of the maleimide type). These functions are generallycarried by a hydrocarbon chain, which is itself joined to the nucleosideor nucleotide part.

Methods of introducing these functional groups are described especiallyin C. Kessler, Nonisotopic probing, Blotting and Sequencing, 2ndedition, L. J. Kricka (1995), published by Academic Press Ltd, London,pp. 66-72.

This spacer arm consists for example of a divalent organic radicalselected from linear or branched C₁-C₂₀ alkylene groups optionallycontaining one or more double bonds or triple bonds and/or optionallycontaining one or more heteroatoms such as oxygen, nitrogen, sulfur orphosphorus, or one or more carbamoyl or carboxamido groups; C₅-C₈cycloalkylene groups; and C₆-C₁₄ arylene groups, said alkylene,cycloalkylene or arylene groups optionally being substituted by alkyl,aryl or sulfonate groups.

In particular, it can be selected from the following groups:

According to a preferred feature, the conjugate according to theinvention comprises deoxyuridine as a deoxyribonucleotide, the europiumcryptate Eu tris-bipyridine as a fluorescent marker and a 3-aminoallylgroup as a spacer arm.

According to another feature, the invention further relates to a processfor the preparation of the conjugates described above.

Said preparative process is characterized in that a ribo- ordeoxyribonucleoside or -nucleotide which is native, chemically modifiedor conjugated with one or more labeling molecules, in which at least onecarbon atom of the ring, exocyclic nitrogen atom of the purine orpyrimidine ring or carbon atom of the pentofuranose unit may be involvedin bonding with a fluorescent marker, is reacted with at least onefluorescent marker bonded to said atom(s) and consisting of a rare earthcryptate, and in that the resulting conjugate is isolated.

The ribo- or deoxyribo-nucleosides or -nucleotides and the fluorescentmarkers which can be used in this preparative process are as describedabove.

The conjugates according to the invention which comprise a ribo- ordeoxyribonucleotide are particularly suitable for all applications whichrequire a qualitative or quantitative measurement during the synthesisor use of polynucleotides.

According to one of its features, the invention further relates to thepolynucleotides comprising at least one fluorescent conjugate of a ribo-or deoxyribonucleotide, as described above, as a constituent nucleotide.

Advantageously, the polynucleotides obtained by the incorporation ofconjugates according to the invention can comprise a number ofconjugates greater than 1 and hence a number of fluorescent markersgreater than 1.

By using the conjugates according to the invention, it is thus possibleto effect the multiple labeling of polynucleotides by an enzymaticmethod. This technique affords labeled polynucleotides more easily andassures a better reproducibility than when using the conventionaltechniques of fluorescent labeling by a chemical method. In particular,it avoids the separation steps which are necessary for removing theunreacted polynucleotides and/or unreacted functionalized fluorescentmarkers from the medium.

It will be noted that it bifunctionalized cryptates are used,conjugation takes place on only one arm, which always allows theincorporation of the conjugate within the framework of a polynucleotidesynthesis.

The conjugates according to the invention which comprise a ribo- ordeoxyribonucleotide can be used for detecting and/or locating compoundscontaining at least one nucleic acid sequence.

Among these uses, the following may be mentioned without implying alimitation:

the detection and/or location of specific DNA sequences, for example formapping chromosomes or detecting a mutation; and

the synthesis of probes which can be used in biomedical research or forestablishing a clinical diagnosis.

They can also be used in a method of measuring the enzymatic activity ofan enzyme involved in a nucleic acid synthesis reaction, for example aDNA or RNA polymerase, reverse transcriptase, transferase or ligaseactivity, where the fluorescence emitted directly or indirectly by saidconjugate is measured, said fluorescence emission being correlated withthe degree of incorporation of said conjugate into the synthesizednucleic acid polynucleotide.

The conjugates according to the invention can also be used for measuringthe enzymatic activity of an enzyme with a nucleic acid substrate, forexample a phosphodiesterase, DNAse or RNAse activity, the fluorescenceemitted directly or indirectly by said conjugate being correlated eitherwith said activity or with the inhibition of said activity.

They can also be used for measuring an enzymatic activity which modifiesthe structure of a nucleic acid, such as a helicase or integraseactivity, or an activity which modifies the topology of a nucleic acid,such as a topoisomerase activity.

The conjugates according to the invention can also be used as markers,for example for the preparation of a compound comprising a nucleic acidinto which said conjugate is incorporated for the purpose of detection.

The fluorescence emitted by the conjugates according to the inventioncan be either “direct”, i.e. the luminous signal is emitted by theconjugate after excitation at a given wavelength, or “indirect”, i.e.the emission of the luminous signal is induced by a non-radiative energytransfer between the excited conjugate, or “donor compound”, and anotherfluorescent molecule, or “acceptor compound”.

In this particular case, the following conditions are fulfilled:

on the one hand, the fluorescent acceptor compound possesses anabsorption spectrum which at least partially overlaps the emissionspectrum of the donor and has a high molar absorbance in this overlapzone, and an emission spectrum over a wavelength range in which thedonor has a weak intrinsic emission;

on the other hand, the acceptor and the donor are situated near oneanother, the orientation of their transition dipoles being approximatelyparallel.

The principle of the technique of non-radiative energy transfer isdescribed especially in G. Mathis et al., Clin. Chem., 1993, 39,1953-1959.

The invention is illustrated by the Examples below, in which someconcentrations are given in absorption units (AU) at a given wavelength(expressed in nm) per unit volume (expressed in ml) and are expressed bythe same number as the optical density of the solution in question.

EXAMPLE 1

Synthesis of Deoxyuridine Labeled With the Cryptate EuropiumTris-bipyridine (Conjugate K-11-dUTP)

In this conjugate the number 11 indicates the total number of atoms inthe spacer arm and in the functional group joining the cryptatestructure to the nucleotide (the bonding in this case takes place in the5-position of the pyrimidine).

The nucleoside triphosphate used is5-[N-(6-aminocaproyl)-3-aminoallyl]-2′-deoxyuridine 5′-triphosphate](AH-dUTP), which is prepared by reacting N-hydroxysuccinimidetrifluoroacetamidocaproate (M. S. Urdea et al., Nucleic Acids Res.,1988, 4937) with 5-(3-aminoalkyl)-2′-deoxyuridine 5′-triphosphate, whichin turn is prepared by a literature process (Langer et al., Proc. Natl.Acad. Sci. USA (1981), 78, 6633-6637), and then carrying out anammoniacal deprotection (3% aqueous NH₄OH, 45 min at 60° C.). Thecompound is purified on DEAE-Sepharose® (Pharmacia) under a lineartriethylammonium hydrogencarbonate gradient (0.1 M to 0.3 M).

1) Method A

68 μl of a solution of AH-dUTP containing 6 μmol/ml (i.e. 0.4 μmol) arediluted with 250 μl of 0.1 M triethylammonium hydrogencarbonate (TEAB),pH 7.8, and 320 μl of a solution of N-hydroxysuccinimide cryptate (4mg/ml in acetonitrile), prepared as below, are added. The cryptateeuropium [(bis-bpy)-(bpy-diester)] described in Example 4, section A, ofpatent application EP 0 321 353 is hydrolyzed with NaOH and the diacidcryptate obtained is purified on an RP-18 HPLC column (gradient ofacetonitrile in 1% aqueous trifluoroacetic acid). The resultingcryptate, europium [(bis-bpy)-(bpy-diacid)] (4 mg), is dissolved in 0.5ml of anhydrous acetonitrile, and 1 mg of N-hydroxysuccinimide is added,followed by a solution of 1.9 mg of dicyclohexylcarbodiimide dissolvedin 0.5 ml of acetonitrile. After a reaction time of 16 h, theprecipitate of dicyclohexylurea is filtered off and the solution ofN-hydroxysuccinimide cryptate is used directly for coupling.

After stirring for 45 min, 15 μl of 1 M TEAB, pH 8.5, are added and themixture is then injected onto a Superdex 30® HR 10/30 column(Pharmacia), which is eluted with 0.05 M TEAB, pH 7, containing 10% ofacetonitrile (flow rate 1 ml/min).

The compound of Rt≡16.4 min is collected and this fraction, calledfraction 1, is then concentrated under vacuum (speed-vac) to a volume of350 μl and contains 8 AU_(304 nm). With an estimated ε₃₀₄ of ≡35,000, itis estimated that the concentration of K-dUTP is about 0.72 mM.

An aliquot (90 μl) of this fraction 1 is injected onto the same column,which is eluted with 25 mM triethylammonium acetate buffer, pH 7,containing 5% of acetonitrile. The fraction corresponding to the onlypeak in the chromatogram is collected (16 min<Rt<19 min) andconcentrated under vacuum (speed-vac) to give 150 μl of a solution ofK-dUTP, called fraction 2, containing 1.95 AU_(304 nm).

The compound is analyzed by mass spectrometry (positive modeelectrospray):

(M−2H)⁺=1431

(M−2H+CH₃COOH)⁺=1491

The UV spectrum in water has a maximum at 241 nm characteristic of thenucleoside part of the molecule (λ_(max)=289 nm, ε=7100; λ_(max)=240 nm,ε=10,700), and a maximum at 304 nm, which is near the λ_(max) of 305 nm(ε=30,000) characteristic of the europium cryptate. A ratio A₃₀₄/A₂₄₁ of≡0.83 is observed, which is compatible with the proposed structure.

2) Method B

0.08 μmol of a solution of AH-dUTP is dissolved in 80 μl of 0.1 M boratebuffer, pH 8.6, and 90 μl of a solution of N-hydroxysuccinimide cryptate(4 mg/ml), prepared as described above in method A, are added.

After 60 min at 20° C., 5 μl of 1 M TEAB, pH 8.5, and 45 μl of H₂O areadded. The whole of the reaction mixture is injected onto a Superdexpeptide HR 10/30 column (Pharmacia), which is eluted with 0.05 M TEAB,pH 7, containing 10% of acetonitrile (flow rate 1 ml/min).

The peak of Rt≡16.1 min, which has a maximum at 304 nm and a ratioA₃₀₄/A₂₄₁ of ≡0.79, is collected to give about 0.03 μmol of the compoundK-11-dUTP.

The formula of the conjugate K-11-dUTP is given in FIG. 2.

EXAMPLE 2

Purification of the Conjugate K-11-dUTP By Ion Exchange

The column used is a C10/20 column (Pharmacia Biotech., Uppsala, S)packed with DEAE Sepharose® Fast Flow (Pharmacia) equilibrated in 10 mMTEAB buffer containing 10% of methanol. A solution of K-11-dUTP, alsocontaining non-conjugated, functionalized europium tris-bipyridinecryptate, is deposited and the column is eluted (8 ml/min) with 40 ml ofbuffer A (10 mM TEAB containing 10% of methanol). 4 ml fractions arecollected and the fluorescence of the eluted fractions is measured(λ_(excitation)=337 nm, λ_(emission)=620 nm). The non-conjugatedcryptate is eluted in fractions 4 and 5, i.e. shortly after the deadvolume of the column.

Elution is continued (8 ml/min) under a linear gradient from 10 mM TEAB,10% methanol (100 ml) to 200 mM TEAB, 10% methanol (100 ml) and 5 mlfractions are collected. It is observed that the fluorescence (620 nm)is concentrated in tubes 43 to 44, indicating that the K-11-dUTP iseluted at a concentration of ≡0.16 mM TEAB. The fractions containing theK-11-dUTP are combined and concentrated.

EXAMPLE 3

Incorporation of the Conjugate K-11-dUTP During the Copying of aSingle-stranded DNA By Means of a Polymerase (Analysis By PAGE andAutoradiography)

The template DNA is obtained by the PCR (Polymerase Chain Reaction)amplification of a fragment of the k-ras gene (exon I) bounded by thefollowing primers:

k-ras EX1 sense S primer ^(5′)d(GGC CTG CTG AAA ATG ACT GAA TAT)_(3′)

stock solution containing 3 AU₂₆₀/ml, i.e. about 1.2.10⁻⁵ M

k-ras EX1 antisense AS primer ^(5′)d(TGT TGG ATC ATA TTC GTC CAC AAAATG)_(3′)

stock solution containing 3 AU₂₆₀/ml, i.e. about 1.2.10⁻⁵ M

The AS primer used here for the PCR below is biotinylated at its 5′ end.A biotinylated double-stranded DNA is synthesized by PCR using thefollowing protocol:

PCR medium 25 μl of BUAM (CIS bio international) 10×

8 μl of antisense-bio primer

8 μl of sense primer

10 μl of Taq polymerase (1.25 U/ml)

10 μl of dNTP (four natural dNTPs, 5 mM each)

100 μl of human placenta DNA (Sigma), 0.01 μg/μl

89 μl of milli-Q water

The PCR medium is divided up into 5 microtubes (5×50 μl) and the tubesare placed in a thermocycler and subjected to 31 PCR cycles according tothe protocol of Example 5.

The double-stranded DNA produced by the PCR (equivalent to 25 pmol ofbiotin primer) is incubated in the presence of 300 μg ofDynabeads-streptavidin M-280 (Dynal, N).

After washing, the single-stranded DNA (SS DNA) is eluted with 0.1 Nsodium hydroxide solution, after which the supernatant is decanted andneutralized (dilute HCl) and then concentrated to a residual volume of60 μl.

An AS primer (non-biotinylated) as defined above, labeled with ³²P, asdescribed in J. Sambrook et al., Molecular cloning, a laboratory manual,1989, is used for the remainder of the manipulation.

Media for the copying reaction are prepared using the following:

K-11-dUTP fraction 1 (Rt=16.4 min) obtained in Example 1, diluted to0.25 mM

dTTP, 0.25 mM

mixture of 3 deoxynucleotide triphosphates (dATP, dCTP and dGTP), 0.625mM each

Taq DNA polymerase, 1.25 U/μl

Taq (BUAM) 10×buffer (Cis bio international)

AS primer (3 AU₂₆₀/ml) and ³²P-labeled AS primer (0.06 AU₂₆₀/ml)

A parallel control reaction is carried out in which the K-11-dUTP isreplaced with 0.6 μl of dTTP (0.25 mM) so that the concentration of dTTPin the medium is the same as for the other three triphosphates.

Volume (μl) BUAM 10X 1 Taq DNA polymerase 0.2 ³²P S primer 2 S primer0.35 3 triphosphates 0.5 K-11-dUTP 0.6 dTTP 0.6 SS DNA 5

Polyacrylamide gel electrophoresis (PAGE) is used to verify that thereaction in the presence of the conjugate K-11-dUTP produces a bandcorresponding to a copy of the DNA over the whole of its length andhaving a mobility close to that of the band obtained for the controlreaction where only the four natural nucleotides are introduced.Furthermore, the electrophoresis profile shows no readingdiscontinuities in either case.

The same manipulation was carried out using variable ratiosdTTP/K-11-dUTP, inter alia with 0.3 μl of K-11-dUTP and 0.9 μl of dTTP(dTTP/K-11-dUTP=3).

The copying reaction was also carried out in the presence of DNApolymerase I, Klenow fragment (37° C., 45 min) to give substantially thesame results.

These results show that the conjugate dUTP/europium tris-bipyridinecryptate is indeed incorporated by a polymerase (Taq polymerase orKlenow fragment) during the copying of the single-stranded DNA.

EXAMPLE 4

Incorporation of the Conjugate K-11-dUTP During the Copying of aSingle-stranded DNA By Means of a Polymerase (Verification ByNon-radiative Energy Transfer and Time Resolved FluorescenceMeasurement)

The single-stranded DNA described in Example 3 is used.

A microtube for carrying out the copying reaction (50 μl) is preparedwhich contains:

K-11-dUTP: 0.25 mM fraction 1 (Rt=16.4 min), obtained in Example 1

dTTP: 0.25 mM

mixture of 3 deoxynucleotide triphosphates: dATP, dCTP and dGTP, 0.625mM each

biotinylated AS primer (cf. Example 3): 3 AU₂₆₀/ml

Taq DNA polymerase: 1.25 U/μl

Taq buffer: BUAM 10×

Volume (μl) BUAM 10X 5 Taq polymerase 1 Biotinylated primer 1.75 Mix of3 dNTps 2.5 K-11-dUTP 3 dTTP 3 SS DNA 30 Milli-Q water 3.75

The tubes are heated for 2 min at 90° C. (denaturation) and thenincubated at 70° C. in a thermocycler. Samples (9 μl) are taken at 0, 5,10, 15 and 20 min. The aliquots are placed in tubes containing 2 μl of0.5 M EDTA solution, pH 8.

This gives the equivalent of 12.5 pmol of biotinylated primers per 50 μlof reaction mixture, i.e. 2.5 pmol per 10 μl. There are also 150 pmol ofK-11-dUTP per 10 μl.

The samples are diluted at a rate of 8 μl in 200 μl of buffer L (0.1 Mphosphate, pH 7, 0.15 M NaCl, 0.4 M KF and 0.1% BSA) and then diluted to1/10 in the same buffer.

20 μl (equivalent to 2.10⁻¹⁴ mol of biotin) of samples (diluted asdescribed above) are pipetted into the “test” wells of a microplate(PACKARD HTRF-96 96-well flat bottom black low fluorescence microplate).30 μl of the conjugate SA-XL₆₆₅ (conjugate of streptavidin andchemically modified allophycocyanin, CIS bio international) diluted inbuffer L (final concentration 2.10⁻⁸ M) are added to each “test” well(6.5.10⁻¹³ mol of SA, i.e. 30 equivalents based on biotin), followed by250 μl of buffer L.

20 μl of diluted samples are pipetted into the “blank” wells, to which280 μl of buffer L are added.

After incubation (15 min at 37° C.), the fluorescence is readimmediately at 620 nm and 665 nm on a Discovery instrument (PACKARD HTRFmicroplate analyzer).

The ratios of the fluorescence intensities, Re=E665/E620 for each testand Ro=B665/B620 for each blank, are calculated and then the value ofDF=(Re−Ro)/Ro, expressed as a percentage, is calculated. The results arereported in Table 1 below.

In the Table, as in the remaining Examples, the fluorescencemeasurements at 620 nm or 665 nm are expressed in arbitrary fluorescenceunits which depend on the instrument used for the measurement.

TABLE 1 Re = Ro = Time E665/ B665/ (min) E665 E620 /E620 B665 B620 B620DF 0 1590 35,770 0.044 1615 39,423 0.041  8% 5 3670 42,016 0.087 171842,961 0.040 118% 10 4077 40,448 0.100 1713 42,234 0.040 149% 15 418538,670 0.108 1633 40,089 0.040 166% 20 5357 49,403 0.108 1993 51,7770.038 182%

A plot of the value of DF as a function of the reaction time gives thetypical profile of the kinetics of nucleotide incorporation duringenzymatic copying (FIG. 1).

EXAMPLE 5

Incorporation of the Conjugate K-11-dUTP During a PCR (Verification byNon-radiative Energy Transfer and Time Resolved FluorescenceMeasurement)

1) PCR

A region of exon 1 of the k-ras gene bounded by the S and AS primersdescribed in Example 3 is amplified.

This generates an amplification product with a length of 117 bp and thefollowing sequence (sense strand):

 ^(5′)d(GGC CTG CTG AAA ¹ATG ACT GAA TAT AAA CTT GTG GTA GTT ¹⁰GGA GCTGGT GGC GTA GGC AAG AGT GCC TTG ²⁰ACG ATA CAG CTA ATT CAG AAT CAT TTTGTG ³⁰GAC GAA TAT GAT CCA ACA)_(3′)

A probe (56% G+C) is selected from the central part of the amplifiedsequence (underlined above). A biotin is introduced at its 5′ end withthe aid of an N-4-aminohexyl-dC arm (A. Roget et al., Nucleic AcidsRes., 1989, 17, 7643-7651): CP probe: biotin-dC.GCC TTG ACG ATA CAG C.

Stock solution containing 0.3 AU₂₆₀/ml, i.e. about 1.7.10⁻⁶ M. 48 μl ofthe K-11-dUTP fraction (13 AU₃₀₄/ml), repurified from a TEAAc buffer(cf. Example 1, method A), are diluted with 32 μl of milli-Q water, i.e.a final concentration of K-11-dUTP of about 0.2 mM.

A mixture of the following 4 natural deoxynucleotide triphosphates isused: 5 mM dATP, 5 mM dCTP, 5 mM dGTP and 3.5 mM dTTP.

The following PCR stock mixture is prepared:

25 μl of BUAM buffer (CIS bio international) 10×

10 μl of dNTP

8 μl of S primer

8 μl of AS primer

10 μl of Taq DNA polymerase (i.e. 12.5 U)

9 μl of milli-Q water

In PCR microtubes, the PCR media are prepared according to the followingTable using 0.01 μg/μl of human placenta DNA (Sigma).

T− T+ K1− K1+ K2− K2+ Mixture (μl) 7 7 7 7 7 7 dTTP (μl) 1.5 1.5 0 0 0 0K-11-dUTP (μl) 0 0 1 1 2 2 DNA (μl) 0 10 0 10 0 10 Milli-Q water (μl)16.5 6.5 17 7 16 6 [K-11-dUTP] (μM) 0 0 8 8 16 16 [dTTP] (μM) 200 200140 140 140 140 [K-11-dUTP]/[dTTP] — — 0.06 0.06 0.12 0.12

The PCR is carried out using the following protocol:

1. 5 min/95° C.,

2.1 1 min/94° C. (denaturation)

2.2 1 min/60° C. (circularization)

2.3 1 min/70° C. (elongation) (31 cycles)

3.1 8 min/70° C. (final elongation)

The reactions are monitored by agarose gel electrophoresis. Only tubescontaining DNA (T+, K1+ and K2+) give a band of the expected length (bycomparison with the 124 bp band of Boehringer marker VIII).

2) Time Resolved Measurement of the Energy Transfer

The principle of the measurement consists in hybridizing thebiotinylated CP probe on the amplified DNA fragment and then bringingthe hybrid into contact with the conjugate SA-XL₆₆₅ (defined in Example4). The incorporation of bases labeled with europium cryptate (donor)results in a non-radiative energy transfer to XL₆₆₅ (acceptor) when thedonor is excited at about 337 nm.

In this case the acceptor emits fluorescence at 665 nm with a longlifetime, enabling this signal to be differentiated from the inherentfluorescence of the acceptor, which has a short lifetime.

Time resolved fluorescence measurements are made at 620 nm and 665 nm inthe presence of the acceptor (“test” E620 and E665) and in the absenceof the acceptor (“blank” B620 and B665) and then the ratios Re=E665/E620and Ro=B665/B620 are calculated. The value of DF=(Re−Ro)/Ro, expressedas a percentage, is then calculated. An increase in the value of DFshows the presence of an energy transfer and hence the incorporation ofthe cryptate into the amplified DNA.

The media K1−, K1+, K2− and K2+ originating from the PCRs are used. 2 μlof each PCR medium are deposited in microtubes, 10 μl of CP probe (0.03AU₂₆₀/ml) and 13 μl of BUAM buffer (2×) are added and the sealed tubesare heated for 10 min at 94° C. and then for 20 min at 50° C. with theaid of a thermocycler. 5 μl of hybridization medium are diluted in 200μl of buffer L, and 40 μl of this dilution are pipetted into the “test”wells of a microtiter plate and 40 μl into the “blank” wells (PackardHTRF 96-well flat bottom microplate).

40 μl of 3.10⁻⁸ M SA-XL₆₆₅ (CIS bio international) and 200 μl of bufferL are added to the “test” wells. 240 μl of buffer L are added to the“blank” wells.

After incubation for 15 min at 37° C., the fluorescence is measuredimmediately on a Discovery instrument (Packard).

The results are reported in Table 2 below.

TABLE 2 Tube E665 E620 Re B665 B620 Ro DF K1− 889 16,053 0.0554  84716,825 0.0503  10% K1+ 2406 28,392 0.0847 1366 32,494 0.0420 102% K2−1320 29,015 0.0455 1273 31,452 0.0405  12% K2+ 3304 36,615 0.0902 149136,278 0.0411 120%

The results show that the conjugate K-11-dUTP is incorporated during aPCR. Furthermore, the presence of the cryptate molecules bonded to theamplified DNA can be demonstrated by hybridization with a probe specificfor this amplified DNA and disclosed by a non-radiative energy transferbetween the cryptate and an acceptor bonded to the hybridized probe.

EXAMPLE 6

Synthesis of Deoxyuridine Labeled With the Cryptate EuropiumTris-bipyridine (K-4-dUTP)

In this compound the number 4 indicates the total number of atoms in thearm joining the cryptate structure to the nucleotide (the bonding inthis case takes place in the 5-position of the pyrimidine).

The nucleoside triphosphate used is 5-(3-aminoallyl)-2′-deoxyuridine5′-triphosphate (AA-dUTP), which is prepared by a literature process(Langer et al., Proc. Natl. Acad. Sci. USA (1981), 78, 6633-6637). Thecompound is purified on DEAE-Sepharose® (Pharmacia) under atriethylammonium hydrogencarbonate gradient (10 mM to 300 mM).

60 μl of a solution of AA-dUTP containing 5.4 μmol/ml (i.e. 0.3 μmol)are diluted with 240 μl of 0.1 M triethylammonium hydrogencarbonate(TEAB), pH 7.8, and 300 μl of a solution of activated cryptate[TBP-(Eu³⁺)] containing 4 mg/ml in acetonitrile, i.e. 0.85 μmol (about 3equivalents), are added. The activated cryptate [TBP-(Eu³⁺)] is preparedfor immediate use as described in Example 1, method A.

After shaking for 35 min, 15 μl of 1 M TEAB, pH 8.5, are added and themixture is concentrated to half and then injected onto a Superdex 30® HR10/30 column (Pharmacia), which is eluted with 25 mM TEAB, pH 9,containing 10% of acetonitrile (flow rate 1 ml/min).

The compound of Rt≡16.3 min is collected; this fraction, called fraction1, is concentrated under vacuum (speed-vac) to a volume of 200 μl andthen injected onto the same column, which is eluted with 25 mMtriethylammonium acetate buffer, pH 7, containing 5% of acetonitrile.The fraction corresponding to the only peak in the chromatogram iscollected (16 min<Rt<19 min) and concentrated under vacuum (speed-vac)to give 260 μl of a solution of K-4-dUTP, called fraction 2, containing6.0 AU₃₀₃. With an estimated ε₃₀₃ of ≡35,000, it is estimated that theconcentration of K-4-dUTP is about 0.65 mM.

The compound is analyzed by mass spectrometry (negative modeelectrospray):

(M−4H)=1315.5 (calculated for C₅₀H₄₈EuN₁₁O₁₇P₃: 1319)

The UV spectrum in water has a maximum at 243 nm characteristic of thenucleoside (λ_(max)=289 nm, ε=7100; λ_(max)=240 nm, ε=10,700), and amaximum at 303 nm, which is near the λ_(max) of 305 nm (ε=27,000)characteristic of the europium cryptate. A ratio A₃₀₃/A₂₄₀ of ≡0.82 isobserved, which is compatible with the proposed structure.

The formula of the conjugate K-4-dUTP is given in FIG. 2.

EXAMPLE 7

Synthesis of Uridine Labeled With the Cryptate Europium Tris-bipyridine(K-11-UTP)

In this compound the number 11 indicates the total number of atoms inthe spacer arm joining the cryptate structure to the nucleotide (thebonding in this case takes place in the 5-position of the pyrimidine).

The nucleoside triphosphate used is6-aminocaproyl-[5-(3-aminoallyl)uridine 5′-triphosphate] (AH-UTP), whichis prepared as indicated in Example 1. The compound is purified onDEAE-Sepharose® (Pharmacia) under a linear triethylammoniumhydrogencarbonate gradient (25 mM to 300 mM).

400 μl of a solution of AH-dUTP containing 1.1 μmol/ml (i.e. 0.44 μmol)in 0.1 M triethylammonium hydrogencarbonate (TEAB), pH 7.8, are used and360 μl of a solution of activated cryptate [TBP-(Eu³⁺)] (3 mg/ml inacetonitrile) are added. The activated cryptate [TBP-(Eu³⁺)] is preparedfor immediate use as described in Example 1, method A.

After stirring for 45 min, 40 μl of 1 M TEAB, pH 8.5, are added and themixture is then injected onto a Superdex peptide 30® HR 10/30 column(Pharmacia), which is eluted with 50 mM TEAB, pH 7, containing 10% ofacetonitrile (flow rate 1 ml/min).

The compound of Rt≡15.4 min is collected; this fraction, called fraction1, is concentrated under vacuum (speed-vac) to a volume of 200 μl, whichcontains ≡10 AU_(304 nm). With an estimated ε₃₀₄ of ≡35,000, it isestimated that the concentration of K-11-UTP is about 0.3 mM.

This fraction 1 is injected onto the same column, which is eluted with25 mM triethylammonium acetate buffer, pH 7, containing 5% ofacetonitrile. The fraction corresponding to the only peak in thechromatogram is collected (Rt≡16.5 min) and concentrated under vacuum(speed-vac) to give 202 μl of a solution of K-11-UTP, called fraction 2,containing 7.5 AU_(304 nm).

The compound is analyzed by mass spectrometry (negative modeelectrospray):

(M−4H)⁻=1444.3 (calculated for C₅₆H₅₈EuN₁₂O₁₉P₃: 1448.0)

The UV spectrum in water has a maximum at 241 nm characteristic of thenucleoside (λ_(max)=289 nm, ε=7100; λ_(max)=240 nm, ε=10,700), and amaximum at 303 nm, which is near the λ_(max) of 305 nm (ε=27,000)characteristic of the europium cryptate. A ratio A₃₀₄/A₂₄₁ of ≡0.80 isobserved, which is compatible with the proposed structure.

The compound is analyzed by FPLC (mono-Q column, Pharmacia). Buffer A:20 mM sodium acetate, pH 5.2, containing 10% of acetonitrile. Buffer B:20 mM sodium acetate, pH 5.0, and 1 M LiCl containing 10% ofacetonitrile. Linear gradient from 0 to 30% B in 25 min. Flow rate 1ml/min. The K-11-UTP has an Rt of 10.7 min.

The formula of the conjugate K-11-UTP is given in FIG. 3.

EXAMPLE 8

Comparative Measurements of the Lifetimes of the Conjugates of dUTP andUTP With the Cryptate Europium Tris-bipyridine and of the Cryptate Alone

The reference cryptate used is the cryptate europium[(bis-bpy)(bpy-di(amidoethyleneamine))] described in Example 4 of patentapplication EP 0 321 353 (hereafter called KNH₂).

A stock solution of KNH₂ in water is prepared and its concentration isdetermined by measurement of the optical density at 306 nm, taking avalue of ≡30,000 for ε₃₀₆. The stock solution (6.7×10⁻⁴ M) is diluted to1/100 in 0.1 M Tris-HCl buffer, pH 9; 100 μl of this intermediatedilution are then diluted in 600 μl of the same buffer. In parallel, astock solution of K-11-dUTP, K-4-dUTP and K-11-UTP (prepared accordingto Example 1, method A, and Examples 6 and 7 respectively) is preparedin water, whose concentration is estimated as 3×10⁻⁵ M by measurement ofthe optical density at 304 nm (ε₃₀₄≡35,000). Each stock solution isdiluted at a rate of 50 μl in 1 ml of 0.1 M Tris-HCl buffer, pH 7.4, orin 0.1 M Tris-HCl buffer, pH 9.

The values of the emission lifetime of the europium (τ in ms) aremeasured using an LS50 time resolved spectrofluorimeter (Perkin-Elmer)and Helma 5 mm×5 mm cells.

The results are reported in Table 3 below:

TABLE 3 Lifetime τ (ms) in 0.1 M Lifetime τ (ms) in 0.1 M Compound Trisbuffer, pH 7.4 Tris buffer, pH 9 K-11-dUTP 0.587 0.596 K-4-dUTP 0.6210.651 K-11-UTP 0.636 0.640 KNH₂ (reference) 0.368 0.265

These results show that the coupling of the nucleotide with the cryptatemolecule results in an increase in the lifetime of the europium and innew fluorescent characteristics for the cryptate/nucleotide conjugaterelative to the reference cryptate.

EXAMPLE 9

Incorporation of K-11-dUTP During the Elongation of an OligonucleotideBy Means of Terminal Nucleotidyl Transferase (Verification ByNon-radiative Energy Transfer and Time Resolved FluorescenceMeasurement)

A microtube for carrying out the elongation reaction is prepared using:

25 μl of 0.2 M Tris-acetate buffer, pH 7.2

4 μl of 25 mM aqueous cobalt chloride solution

5 μl of a 2.4 μM solution of an oligonucleotide (composition A₂C₃G₇T₃biotinylated at the 5′ end) in H₂O

2 μl of K-11-dUTP (fraction 2, prepared according to Example 7) at aconcentration 0.04 mM

8 μl of 0.04 mM dTTP

5 μl of water

1 μl of terminal deoxynucleotidyl transferase (TnT, EC 2.7.7.31) (Sigma,35 U/μl)

2 μl of the reaction mixture are taken and added to 4 μl of 60 mM EDTA(to obtain a control at t₀=0 min) and the remainder is incubated at 37°C. for a kinetic study. After reaction times of 5, 10, 20, 40, 60 and 80min at 37° C., 2 μl of the reaction mixture are taken and added to 4 μlof 60 mM EDTA in order to stop the reaction. This gives fractions t₅,t₁₀ etc. These fractions t₀, t₅, t₁₀ are diluted with 250 μl of buffer L(0.1 M phosphate, pH 7, 0.15 M NaCl, 0.4 M KF and 0.1% BSA).

50 μl (equivalent to 2.3.10⁻¹² mol of biotin) of samples diluted asdescribed above are pipetted into the “test” wells (called E₀, E₅, E₁₀etc.) of a microplate (PACKARD HTRF-96 96-well flat bottom black lowfluorescence microplate). 50 μl of the conjugate SA-XL₆₆₅ (CIS biointernational) diluted in buffer L (1.5.10⁻⁸ M) are added to each “test”well, followed by 150 μl of buffer L.

A “blank” is prepared from a reaction mixture described above in whichthe enzyme has been replaced with 1 μl of water. 2 μl of this mixtureare taken and subjected to the treatments and dilutions described above.50 μl of this diluted sample are pipetted into the “blank” wells, towhich 50 μl of the conjugate SA-XL₆₆₅ and 150 μl of buffer L are added.

After incubation (15 min at 37° C.), the fluorescence is read at 620 nmand 665 nm on a DISCOVERY instrument (PACKARD HTRF microplate analyzer).

The ratios of the fluorescence intensities, Re=E665/E620 for each testand Ro=B665/B620 for each blank, are calculated and then the value ofDF=(Re−Ro)/Ro, expressed as a percentage, is calculated. The results arereported in Table 4 below.

TABLE 4 Time (min) 0 5 10 20 40 60 80 F665 602 1585 2186 3610 5347 82045542 F620 9559 10,757 9921 11,023 10,754 10,589 8971 Re = 0.063 0.1470.22 0.327 0.497 0.77 0.618 F665/F620 DF 0 129 242 409 673 1104 865

The blank has a ratio B₆₆₅/B₆₂₀ of 0.064. For t₀ a ratio E₆₆₅/E₆₂₀ of0.063 is observed, the transfer beingDF=100×(Re−Ro)/Ro=100(0.063-0.064)/0.064, i.e. virtually zero. Bycalculating DF for each time in this way, it is observed that the energytransfer expressed as the value of DF increases over time, showing thatthe K-11-dUTP is incorporated into the oligonucleotide chain by theterminal nucleotidyl transferase.

EXAMPLE 10

Photophysical Properties of an Oligonucleotide into Which CryptateMolecules are Incorporated

A reaction to incorporate K-11-dUTP by means of TnT is carried outaccording to Example 9 with a 3-fold increase in all the amounts. DF ismeasured as a function of time in order to verify that the incorporationof K-11-dUTP follows the same kinetics. After a reaction time of 80 min,the reaction is stopped with 12 μl of 400 mM EDTA solution. 105 μl ofthe reaction mixture are taken and deposited on an NAP5 column(Pharmacia) equilibrated in 10 mM phosphate buffer, pH 7.4.

The oligonucleotide is eluted with 600 μl of buffer. This fraction,called “fraction 1” (exclusion volume), is analyzed as follows: 50 μl offraction 1 are deposited in the wells of a microplate and 200 μl ofbuffer L are added to give the “cryptate blank”. 50 μl of fraction 1, 50μl of the conjugate SA-XL₆₆₅ (CIS biointernational) (1.5.10⁻⁸ M inbuffer L) and 150 μl of buffer L are deposited in the adjacent “test”wells. The fluorescence is measured at 620 nm and 665 nm on a DISCOVERYinstrument (PACKARD HTRF microplate analyzer).

The lifetime τ is calculated from the slope of the line obtained byplotting log E₆₂₀=f(t).

The emission lifetime is measured at 620 nm for the labeledoligonucleotide contained in fraction 1 (10 mM phosphate buffer, pH 7.4)using a KRYPTOR time resolved fluorimeter (CIS bio international) withexcitation at 337 nm. This gives a lifetime τ of 866 μs. This lifetimeis longer than that of the conjugate K-11-dUTP (τ=681 μs), measuredsimultaneously under the same conditions.

By comparing the fluorescence of fraction Fl (which corresponds to thecryptate molecules incorporated into the oligonucleotide chain) with thefluorescence of standard solutions containing a known concentration ofcryptate, it is possible to estimate that fraction Fl contains about5.10⁻¹¹ mol of cryptate, given that the estimated amount ofoligonucleotide in fraction Fl is 2.10⁻¹¹, it is observed that a numbern=5.10⁻¹¹/2.10⁻¹¹=2.5 labeled cryptate-labeled nucleotides have beenincorporated on average per oligonucleotide chain.

In this compound the number 8 indicates the total number of atoms in thespacer arm joining the cryptate structure to the nucleotide (the bondingin this case takes place in the 8-position of the purine).

The nucleoside triphosphate used is [8-(6-aminohexyl)adenosine5′-triphosphate] (AH-ATP, Sigma). A solution of 0.1 μmol of AH-ATP in100 μl of 0.1 M triethylammonium hydrogencarbonate (TEAB), pH 8, is usedand 100 μl of a solution of activated cryptate [TBP-(Eu³⁺)] (4 mg/ml inacetonitrile) are added. The activated cryptate [TBP-(Eu³⁺)] (NHS/DCC inacetonitrile) is prepared for immediate use as described in Example 1,method A.

After shaking for 35 min, 5 μl of 1 M TEAB are added and the mixture isconcentrated to half and then injected onto a Superdex 30® HR 10/30column (Pharmacia), which is eluted with 50 mM TEAB, pH 8, containing10% of acetonitrile (flow rate 1 ml/min).

The compound of Rt≡16.7 min is collected; this fraction, called fraction1, is concentrated under vacuum (speed-vac) to a volume of 200 μl andthen injected onto the same column, which is eluted with 25 mMtriethylammonium acetate buffer, pH 7, containing 5% of acetonitrile.The fraction corresponding to the only peak in the chromatogram iscollected (Rt=17.2 min) and concentrated under vacuum (speed-vac).

The UV spectrum in water has a maximum at 245 nm, a maximum at 283 nm,which is near the λ_(max) characteristic of the nucleoside (λ_(max)=279,ε=21,000), and a maximum at 303 nm, which is near the λ_(max) of 305 nm(ε=27,000) characteristic of the europium cryptate. A ratio A₃₀₅/A₂₈₀ of≡1 is observed, which is compatible with the proposed structure.

The formula of the conjugate K-8-ATP is given in FIG. 3.

EXAMPLE 12

Incorporation of a Conjugate of UTP With the Cryptate EuropiumTris-bipyridine (K-11-UTP) During an In Vitro Transcription Reaction(Verification By Non-radiative Energy Transfer and Time ResolvedFluorescence Measurement)

This Example concerns the simultaneous incorporation of K-11-UTP andbio-14-CTP (CTP/biotin conjugate) into one and the same RNA molecule byin vitro transcription.

1/Transcription

The transcription reaction of a double-stranded DNA containing apromoter (plasmid DNA pSTP18 and pSTP19 containing the T7 promoter andlinearized by EcoRI) to RNA is carried out with the aid of an SP6/T7transcription kit (Boehringer-Mannheim). The transcript obtained has alength of 1035 bases.

In addition to the ribonucleotides at a final concentration of 0.5 mM,the transcription medium contains 0.2% of K-11-UTP and 30% ofbio-14-CTP, which will be incorporated randomly along the chain.

The K-11-UTP, fraction 2, described in Example 7 is diluted in water togive a concentration of 20 μM.

The solution of bio-14-CTP is prepared by diluting a commercial 10 mMsolution of biotin-14-CTP (Gibco-BRL/Life Technologies) in water.

The transcription medium is prepared in PCR microtubes (0.5 ml)according to the Table below: bio-14-CTP (CTP/biotin conjugate) into oneand the same RNA molecule by in vitro transcription.

1/Transcription

The transcription reaction of a double-stranded DNA containing apromoter (plasmid DNA pSTP18 and pSTP19 containing the T7 promoter andlinearized by EcoRI) to RNA is carried out with the aid of an SP6/T7transcription kit (Boehringer-Mannheim). The transcript obtained has alength of 1035 bases.

In addition to the ribonucleotides at a final concentration of 0.5 mM,the transcription medium contains 0.2% of K-11-UTP and 30% ofbio-14-CTP, which will be incorporated randomly along the chain.

The K-11-UTP, fraction 2, described in Example 7 is diluted in water togive a concentration of 20 μM.

The solution of bio-14-CTP is prepared by diluting a commercial 10 mMsolution of biotin-14-CTP (Gibco-BRL/Life Technologies) in water.

The transcription medium is prepared in PCR microtubes (0.5 ml)according to the Table below:

Labeled rNTP/ Final labeled rNTP + Volume concentration natural rNTPTranscription buffer (10X) 2 μl Plasmid DNA (T7) (0.5 μg/μl) 2 μl ATP (5mM) 2 μl 0.5 mM GTP (5 mM) 2 μl 0.5 mM UTP (5 mM) 2 μl 0.5 mM K-11-UTP(20 μM) 2 μl 2 μM 0.4% CTP (3.5 mM) 2 μl 0.35 mM Bio-14-CTP (1.5 mM) 2μl 0.15 mM 30% RNAse inhibitor (20 U/μl) 1 μl H₂O 1 μl T7 RNA polymerase2 μl Total volume 20 μl 

After addition of the enzyme, 2 μl of the reaction medium are taken andimmediately diluted with 38 μl of 50 mM EDTA solution, pH 8. Thissolution is then diluted by taking 19 μl and diluting it with 114 μl ofbuffer L. This solution will be used as a background control at t₀=0min.

The reaction mixture is placed in a thermocycler at 37° C. After givenreaction times (60 min, 90 min and 120 min), 2 μl of the reactionmixture are taken and diluted, as previously, with 38 μl of 50 mM EDTA,pH 8. These solutions are then diluted by taking 19 μl and diluting itwith 114 μl of buffer L.

50 μl of each diluted solution, corresponding to the times 0 min, 60min, 90 min and 120 min, are deposited in the wells of a blackmicroplate (Packard HTRF 96-well flat bottom microplate).

50 μl of a solution of SA-XL₆₆₅ (CIS bio international) at aconcentration of 6.25.10⁻⁷ M in buffer L are added, followed by 100 μlof buffer L.

2/ Time Resolved Measurement of the Energy Transfer

After incubation (15 min at room temperature), the fluorescence ismeasured at 620 nm and 665 nm on a DISCOVERY instrument (Packard).

For each reaction time considered, the energy transfer is evaluated bycalculating the ratio Re=F665/F620. The ratio Ro=F665/F620 for the timet₀ makes it possible to evaluate the background level. The samemeasurement made on a dilution of 2 μl of a transcription reactionmixture in which the enzyme has been replaced with an equivalent volumeof water gives the same background level.

The energy transfer is calculated by the formula DF=(Re−Ro)/Ro for eachtime. The results are reported in Table 5 below.

TABLE 5 Time DF = (Re − Ro)/Ro (min) F665 F620 Re = F665/F620 (%) 0 210814,672 0.143 0 60 6329 18,755 0.337 135 90 6672 17,026 0.392 174 1207386 18,179 0.406 184

It is observed that the value of DF increases, representing an increasein the energy transfer due to the incorporation of K-11-UTP and bio-CTPinto the transcribed RNA.

The transcription reaction described above is monitored byelectrophoresis on agarose gel (3%).

A large band is observed, showing the integrity of the RNA fragmentproduced. This band is characterized by an identical migration to thatof the band produced from a transcription performed on the natural rNTPsalone.

The curve of the values of DF as a function of reaction time, given inFIG. 4, shows the typical profile of the kinetics of nucleotideincorporation during an enzymatic transcription. These results show thatthe conjugate K-11-UTP is incorporated efficiently by an RNA polymerase.

EXAMPLE 13

Incorporation of a Conjugate K-11-dUTP During an In Vitro TranscriptionReaction (Verification By Non-radiative Energy Transfer and TimeResolved Fluorescence Measurement After Hybridization With an AcceptorProbe)

The transcription reaction of a double-stranded DNA to RNA is carriedout with the aid of a transcription kit (Gibco BRL). The double-strandedDNA containing the T7 promoter is itself obtained by a PCR carried outwith a pair of primers, one of which contains the sequence of thepromoter of T7 RNA polymerase (EC 2.7.7.6).

The transcript obtained has a theoretical length of 115 bases.

The general principle of transcription by an RNA polymerase is describedin p. 5.58 and 5.59 of “Molecular Cloning, A Laboratory Manual”, 2ndedition, J. Sambrook, E. F. Fritsch and T. Maniatis, CSH Press 1989.

It is also possible to use another RNA polymerase, such as SP6 or T3 RNApolymerase, in which case the sequence of the PCR primer in questionwill be modified in order to incorporate the specific promoter of theRNA polymerase considered.

The incorporation of a promoter is described in §13.5 and §23.2 of “PCR:Clinical Diagnostics and Research”, A. Rolfs et al., Springer-Verlag(1992), and in D. Y. Kwoth et al., Proc. Natl. Acad. Sci. USA (1989),86, 1173-1177.

1/Preparation of the Double-stranded DNA Containing the Promoter of RNAPolymerase

The protocol used is analogous to that described in Example 5 exceptthat only deoxynucleotides are used to generate a primary PCR product of117 bp.

A second PCR (secondary PCR) is carried out using 2 μl of a 1/100dilution of the product of the primary PCR as the target DNA (DNA). Forthis secondary PCR the k-ras EX1 antisense primer is replaced with aprimer, called T7-AS, of the following sequence:

^(5′)d(TAA TAC GAC TCA CTA TAG GGG TGG ATC ATA TTC GTC CAC AAA ATG)_(3′)

The italicized part of this sequence corresponds to the sequence of thepromoter of T7 RNA polymerase.

The k-ras EX1 sense primer has the following sequence:

^(5′)d(CTG.CTG.AAA.ATG.ACT.GAA.TAT)_(3′)

The double-stranded DNA obtained after this secondary PCR has atheoretical length of 132 bp and the following sequence (sense strand):

^(5′)d(CTG.CTG.AAA.ATG.ACT.GAA.TAT.AAA.CTT.GTG.GTA.GTT.GGA.GCT.GGT.GGC.GTA.GGC.AAG.AGT.GCC.TTG.ACG.ATA.CAG.CTA.ATT.CAG.AAT.CAT.TTT.GTG.GAC.GAA.TAT.GAT.CCA.CCC.CTA.TAG.TGA.GTC.GTA.TTA)_(3′)

The italicized part represents the T7 sequence (sense strand) and theunderlined part represents the sequence corresponding to the probeduring the hybridization step.

Transcription will produce the following RNA sequence (sequencehomologous to the antisense strand of the transcribed DNA fragment), inwhich the underlined part corresponds to the sequence recognized by thebiotinylated probe:

^(5′)pppG.UGG.AUC.AUA.UUC.GUC.CAC.AAA.AUG.AUU.CUG.AAU.UAG.CUG.UAU.CGU.CAA.GGC.ACU.CUU.GCC.UAC.GCC.ACC.AGC.UCC.AAC.UAC.CAC.AAG.UUU.AUA.UUC.AGU.CAU.UUU.CAG.CAG.GCC_(3′)

The RAS12N probe used is biotinylated at the 5′ end and has thefollowing sequence:

biotin-^(5′)d(GTT.GGA.GCT.GGT.GGC.GTA.GG)_(3′)

2/Transcription

In addition to the natural ribonucleotides at a final concentration of0.5 mM, the transcription medium contains 2% of K-11-UTP, which will beincorporated randomly along the RNA chain.

The K-11-UTP, fraction 2, described in Example 7 is diluted in water togive a concentration of 100 μM.

The transcription medium is prepared in PCR microtubes (0.5 ml)according to the Table below:

Final K-11-UTP/ Volume concentration (K-11-UTP + UTP) Transcriptionbuffer(5X) 10 μl Positive PCR DNA(T7) 25 μl (0.5 μg/μl) DTT, 0.1M   2 μlMix of rNTPs (5 mM   5 μl 0.5 mM each) K-11-UTP (100 μM)   5 μl 10 μM 2%RNAse inhibitor(20   2 μl U/μl) H₂O 0.5 μl  T7 RNA polymerase(50 0.5 μl U/μl) Total volume 50 μl

The reaction mixture is placed in a thermocycler at 40° C. for 120 minand the reaction is then stopped with 4 μl of 0.2 M EDTA, pH 8.

2 μl of the reaction medium are placed in a PCR microtube and dilutedwith 10 μl of the RAS12N probe (0.03 AU₂₆₀/ml in H₂O) and 13 μl ofhybridization buffer (100 mM phosphate buffer, pH 7.4, 0.1% BSA, 1 MNaCl) are then added. The mixture is heated for 10 min at 70° C. andthen hybridized for 20 min at 45° C. in a thermocycler.

5.5 μl of the above hybridization medium are diluted in buffer L (qsp200 μl) to give the diluted hybridization medium HT_(pos).

A blank is prepared by performing a transcription according to the aboveprotocol except that the positive PCR DNA is replaced with an equivalentvolume of PCR medium originating from a negative PCR, which does notcontain the target DNA.

The negative transcription medium (2 μl) is stopped, a hybridization isthen carried out as described above and the mixture is diluted in bufferL to give the diluted hybridization medium HT_(neg).

3/ Time Resolved Measurement of the Energy Transfer

The incorporation of the cryptate into the RNA chain is demonstrated byhybridization with a probe complementary to the RNA sequence, this probebeing labeled with the conjugate SA-XL₆₆₅ (CIS bio international) viathe pair streptavidin/biotin.

50 μl of the HT_(pos) medium and 50 μl of the conjugate SA-XL₆₆₅ (CISbio international) diluted to 1.5.10⁻¹⁰ M in buffer L are deposited inthe wells of a black microplate (Packard HTRF 96-well flat bottommicroplate) and 100 μl of buffer L are then added. These wells willenable the energy transfer to be measured by calculating Re=E665/E620 asdetailed in Example 5.

50 μl of the HT_(neg) medium are deposited in the adjacent wells and 50μl of the conjugate SA-XL₆₆₅ and 100 μl of buffer L are added asdescribed above. These wells serve as a reference for evaluating thebackground by calculating Ro.

The fluorescence is measured at 620 nm and 665 nm on a DISCOVERYinstrument (Packard).

The energy transfer is calculated by means of the formula DF=(Re−Ro)/Rofor each time. The results are reported in Table 6 below.

TABLE 6 Medium E665 E620 Re = E665/E620 DF (%) HT_(neg)  738 17,9000.041 HT_(neg) 1714 15,010 0.114 178

A transfer of 178% is observed in the case where the transcription isperformed in the presence of the target DNA.

8 1 24 DNA Artificial Sequence Description of Artificial SequencePRIMER1 ggcctgctga aaatgactga atat 24 2 27 DNA Artificial Sequence Descriptionof Artificial SequencePRIMER 2 tgttggatca tattcgtcca caaaatg 27 3 117DNA Artificial Sequence Description of Artificial SequencePRIMER 3ggcctgctga aaatgactga atataaactt gtggtagttg gagctggtgg cgtaggcaag 60agtgccttga cgatacagct aattcagaat cattttgtgg acgaatatga tccaaca 117 4 16DNA Artificial Sequence Description of Artificial SequencePRIMER 4gccttgacga tacagc 16 5 45 DNA Artificial Sequence Description ofArtificial SequencePRIMER 5 taatacgact cactataggg gtggatcata ttcgtccacaaaatg 45 6 132 DNA Artificial Sequence Description of ArtificialSequencePRIMER 6 ctgctgaaaa tgactgaata taaacttgtg gtagttggag ctggtggcgtaggcaagagt 60 gccttgacga tacagctaat tcagaatcat tttgtggacg aatatgatccacccctatag 120 tgagtcgtat ta 132 7 115 RNA Artificial SequenceDescription of Artificial SequencePRIMER 7 guggaucaua uucguccacaaaaugauucu gaauuagcug uaucgucaag gcacucuugc 60 cuacgccacc agcuccaacuaccacaaguu uauauucagu cauuuucagc aggcc 115 8 20 DNA Artificial SequenceDescription of Artificial SequencePRIMER 8 gttggagctg gtggcgtagg 20

What is claimed is:
 1. A fluorescent conjugate of a nucleoside ornucleotide, comprising: a ribo- or deoxyribo-nucleoside or -nucleotidewhich is native, chemically modified or conjugated with one or morelabeling molecules, in which either: (a) at least one carbon atom of thering or exocyclic nitrogen atom of the purine or pyrimidine ring or ofthe carbon atom of the pentofuranose unit is able to bond with afluorescent marker; or (b) at least one carbon atom of the ring orexocyclic nitrogen atom of the purine or pyrimidine ring is able to bondwith a fluorescent marker; and at least one fluorescent markerconsisting of a rare earth cryptate bonded to said atom(s).
 2. Aconjugate according to claim 1, wherein the ribo- or deoxyribonucleotideis selected from the group consisting of: 2′-deoxyuridine5′-triphosphate or uridine 5′-triphosphate derivatives functionalized inthe 5-position of the base; 2′-deoxycytidine 5′-triphosphate or cytidine5′-triphosphate derivatives functionalized in the 4- or 5-position ofthe base; 2′-deoxyadenosine 5′-triphosphate or adenosine 5′-triphosphatederivatives functionalized in the 6- or 8-position of the base;2′-deoxyguanosine 5′-triphosphate or guanosine 5′-triphosphatederivatives functionalized in the 6- or 8-position of the base;2′-deoxy-7-deazaadenosine 5′-triphosphate or 7-deazaadenosine5′-triphosphate derivatives functionalized in the 7-position of thebase; and 2′-deoxy-7-deazaguanosine 5′-triphosphate or 7-deazaguanosine5′-triphosphate derivatives functionalized in the 7-position of thebase.
 3. Conjugate according to claim 1, characterized in that it is afluorescent conjugate of a nucleotide comprising a ribonucleotideselected from AMP, ADP, ATP, GMP, GDP, GTP, CMP, CDP, CTP, UMP, UDP,UTP, TMP, TDP, TTP, 2Me-AMP, 2Me-ADP, 2Me-ATP, 1Me-GMP, 1Me-GDP,1Me-GTP, 5Me-CMP, 5Me-CDP, 5Me-CTP, 5MeO-CMP, 5MeO-CDP, 5MeO-CTP,7-deaza-ATP and 7-deaza-GTP, or a deoxyribonucleotide selected from thedeoxy- or dideoxyribonucleotides corresponding to these ribonucleotides.4. A conjugate according to claim 1, which is a fluorescent conjugate ofa nucleoside in which the ribo- or deoxyribonucleoside is selected fromthe group consisting of A, G, C, U, T, the corresponding deoxy- ordideoxynucleosides and their chemically modified analogs.
 5. A conjugateaccording to claim 1, wherein the deoxyribonucleoside ordeoxribonucleotide is selected from the group consisting of3′-azido-3′-deoxythymidine, derivatives of 3′-azido-3′-deoxythymidineand the 2′,3′-dideoxy analogs of A, T, C, G, U and I.
 6. A conjugateaccording to claim 1, wherein the fluorescent marker is bonded to afunctional group introduced onto or generated on the base or on thepentofuranose unit of the ribo- or deoxyribo-nucleoside or -nucleotide,either directly or via a spacer arm.
 7. A conjugate according to claim1, wherein the fluorescent marker is selected from the group consistingof a terbium, europium, samarium and dysprosium cryptate.
 8. A conjugateaccording to claim 1, wherein the fluorescent marker is a rare earthcryptate consisting of at least one rare earth salt completed by amacropolycyclic compound of the formula

in which Z is a tri- or tetravalent atom, R is nothing, hydrogen, thehydroxyl group, an amino group or a hydrocarbon radical, and thedivalent radicals Â, {circle around (B)} and Ĉ independently of oneanother are hydrocarbon chains which optionally contain one or moreheteroatoms and are optionally interrupted by a heteromacrocycle, atleast one of the radicals Â, {circle around (B)} and Ĉ also containingat least one molecular moiety or essentially consisting of a molecularmoiety, said molecular moiety possessing a greater triplet energy thanthat of the emission level of the completed rare earth ion.
 9. Aconjugate according to claim 8, wherein the fluorescent marker is a rareearth cryptate of formula (I) in which the molecular moiety is selectedfrom the group consisting of phenanthroline, anthracene, benzene,naphthalene, bi- and terphenyl, azobenzene, azopyridine, pyridine,bipyridines, bis-quinolines and the compounds of the following formulae: —C₂H₄—X₁—C₆H₄—X₂—C₂H₄— —C₂H₄—X₁—CH₂—C₆H₄—CH₂—X₂—C₂H₄— in which X₁ andX₂, which can be identical or different, are oxygen, nitrogen or sulfur,and

in which X is oxygen or hydrogen.
 10. A conjugate according to claim 8,wherein the fluorescent marker is a rare earth cryptate consisting of aterbium or europium ion complexed by a macrocyclic compound selectedfrom the group consisting of: [2.2.phenanthroline]; [2.2.phenanthrolineamide]; [2.2.anthracene]; [2.2.anthracene amide]; [2.2.biisoquinoline];[2.2.biphenyl-bis-pyridine]; [2.2.bipyridine]; [2.2.bipyridine amide];and tris-bipyridine, tris-phenanthroline, phenanthroline-bisbipyridine,biisoquinoline-bis-bipyridine and bis-bipyridine diphenylbipyridinemacropolycycles.
 11. A conjugate according to claim 10, wherein thefluorescent marker is the europium cryptate Eu tris-bipyridine.
 12. Aconjugate according to claim 1, wherein the fluorescent marker is a rareearth cryptate consisting of at least one rare earth salt complexed by amacropolycyclic compound comprising a molecular moiety selected from thegroup consisting of bipyrazines, bipyrimidines and nitrogen heterocyclescontaining N-oxide groups.
 13. A conjugate according to claim 1, whereinthe fluorescent marker is a rare earth cryptate consisting of at leastone rare earth salt complexed by a macropolycyclic compound of formulaII or III below:

in which: the ring of the formula

 is one of the following rings:

Y is a group or spacer arm consisting of a divalent organic radicalselected from linear or branched C₁, to C₂₀ alkylene groups optionallycontaining one or more double bonds and/or optionally containing one ormore heteroatoms such as oxygen, nitrogen, sulfur or phosphorus, or oneor more carbamoyl or carboxamido groups, from C₅ to C₈ cycloalkylenegroups or from C₆ to C₁₄ arylene groups, said alkylene, cycloalkylene orarylene groups optionally being substituted by alkyl, aryl or sulfonategroups; Z is a functional group capable of bonding covalently with abiological substance; R is a methyl group or the group —Y—Z; and R′ ishydrogen or a group —COOR″, in which R″ is a C₁ to C₁₀ alkyl group, orR′ is a group —CO—NH—Y—Z.
 14. A conjugate according to claim 1, whereinthe fluorescent marker is bonded to the ribo- or deoxyribo-nucleoside or-nucleotide via a spacer arm consisting of a divalent organic radicalselected from linear or branched C₁-C₂₀ alkylene groups optionallycontaining one or more double bonds or triple bonds and/or optionallycontaining one or more heteroatoms selected from the group consisting ofoxygen, nitrogen, sulfur and phosphorus, or one or more carbamoyl orcarboxamido groups; C₅-C₈ cycloalkylene groups; and C₆-C₁₄ arylenegroups, said alkylene, cycloalkylene or arylene groups optionally beingsubstituted by alkyl, aryl or sulfonate groups.
 15. A conjugateaccording to claim 14, wherein the spacer arm is selected from thefollowing groups:


16. A conjugate according to claim 6, wherein the deoxyribonucleotide isdeoxyuridine, the fluorescent marker is the europium cryptate Eutris-bipyridine and the spacer arm is a 3-aminoalkyl group.
 17. Aprocess for preparing the conjugate according to claim 1, wherein aribo- or deoxyribo-nucleoside or -nucleotide which is native, chemicallymodified or conjugated with one or more labeling molecules, in which atleast one carbon atom of the ring or exocyclic nitrogen atom of thepurine or pyrimidine ring or else carbon atom of the pentofuranose unitis able to bond with a fluorescent marker, is reacted with at least onefluorescent marker consisting of a rare earth cryptate bonded to saidatom(s).
 18. A polynucleotide comprising more than one conjugateaccording to claim
 8. 19. A method of measuring the activity of anenzyme involved in a nucleic acid synthesis reaction, comprisingpreparing a fluorescent conjugate of a nucleoside or nucleotide,comprising: a ribo- or deoxyribo-nucleoside or -nucleotide which isnative, chemically modified or conjugated with one or more labelingmolecules, in which either (a) at least one carbon atom of the ring orexocyclic nitrogen atom of the purine or pyrimidine ring or of thecarbon atom of the pentofuranose unit is able to bond with a fluorescentmarker; or (b) at least one carbon atom of the ring or exocyclicnitrogen atom of the purine or pyrimidine ring is able to bond with afluorescent marker; and at least one fluorescent marker consisting of arare earth cryptate bonded to said atom(s); carrying out the nucleicacid synthesis reaction using said conjugate as a constituent nucleotidein the reaction; and measuring fluorescence emitted directly orindirectly by the conjugate; wherein the enzymatic activity is indicatedby the amount of fluorescence emitted.
 20. A method according to claim9, wherein the enzyme is selected from the group consisting of DNA orRNA polymerase, reverse transcriptase, transferase and ligase.
 21. Amethod according to claim 19, comprising measuring enzymatic activity inthe presence of a nucleic acid substrate.
 22. A conjugate according toclaim 13, wherein R″ is selected from the group consisting of methyl,ethyl and tert-butyl.